There is concern that chronic exposures from low doses of pesticides over time can lead to negative health outcomes, such as cancers, neurobehavioral deficits in children from in utero exposure and in adults from occupational exposure, and chronic bronchitis. The people do not show acute symptoms, but may have a harmful biological effect. Therefore, development of sensitive and rapid analytical methods for measurement of sub-clinical exposures to pesticides is of particular interest of the National Institute for Occupational Safety and Health (NIOSH), the sector of pesticide illness & injury surveillance in National Occupational Research Agenda (NORA). Agricultural workers, groundskeepers, pet groomers, fumigators, and a variety of other occupations are at risk for exposure to pesticides including fungicides, herbicides, insecticides, rodenticides, and sanitizers. Once validated, this device can readily be employed to on-site assess/screen health risks of occupational pesticides exposure, especially subclinical level, and mitigate the adverse health effects. Research to Practice (r2p). The state-of-the-art scientific findings and our technical know-how will be incorporated and integrated to make a microanalytic device, providing an early warning system of any harmful effects in work related diseases and injuries due to pesticide exposures. Cholinesterase (ChE) enzyme activity has historically been a key biomarker for assessing the health risk of occupational exposure to organophosphorus (OP) and carbamate pesticides. However, the major challenge with this assay is the need of a baseline activity determination prior to the exposure. Since the pre-exposure ChE activity in a general population is obviously not measured in advance of an exposure and the individual's ChE activity fluctuates over time, current method is not accurate and may provide ambiguous results at subclinical exposure. We will develop a new integrated approach and sensor device for accurate biomonitoring of low level exposure to pesticides by using both enzyme inhibition and phosphorylated ChE adducts (OP-ChE) as biomarkers. This will be done by parallel measurements of ChE enzyme activity (thiocholine formation) and total enzyme concentration (by electrochemical immunoassay) from a post-exposure human blood sample instead of pre-exposure one. Based on this approach, a field-deployable microanalytical device with improved sensitivity will be developed for rapid and in-field detection of pesticides exposure. This method uses the total amount of ChE enzyme from a post-exposure sample as individual baseline, which is better than pre-exposure enzyme activity for inhibition calculation because an individual's level of pre-exposure ChE activity fluctuates over time. Such an integrated approach will provide the results of dual biomarkers of enzyme inhibition and inhibited enzyme adducts, therefore, it will be more accurate and reliable compared with other methods which only use one biomarker (activity or OP-ChE). In this proposed work, model pesticides (chlorpyrifos, diazinon, and carbaryl) will be used for demonstration. The test strip-based microanalytical device will be developed and statistically validated with in vitro human blood samples dosed with a range of pesticide concentrations of relevance to occupational exposures. Finally, the device will be statistically validated with human blood samples collected from agricultural workers with various pesticide exposure levels. For development of immunochromatographic/electrochemical sensor, apoferritin-templated inorganic nanoparticles such as CdS will be prepared as labels for amplified electrochemical detection of ChE in blood samples with an integrated electrochemical detector. Graphene or carbon nanotube will be used as electrode-modifying materials for enhancing the sensitivity of enzyme activity assay.